Prior assay techniques for detecting ligands in a specimen have utilized the strong but noncovalent interaction between biotin and the highly basic protein avidin. In one such assay, described by Guesdon et al., J. Histochem. Cytochem. 27, 8:1131-1139 (1979), known as the Bridged Avidin-Biotin (BRAB) technique, a test sample such as serum containing an unknown antigen or antibody is reacted with a solid phase coated with the corresponding antibody or antigen. The test sample is then reacted with a biotinylated protein form of the same antigen or antibody used to coat the bead. This "sandwich" is then reacted with free avidin and a biotin-labeled indicator enzyme. The enzyme activity measured is directly proportional to the amount of unknown antigen or antibody present in the serum sample.
The BRAB technique described above has several limitations. The highly basic avidin carries a high positive charge and can nonspecifically adsorb to any negatively-charged biological components in the assay. The catalytic activity of the indicator enzymes can be destroyed or reduced upon conjugation to biotin. Also, this method can not be easily adapted to inhibition-type assays because of steric considerations.
A variation of the BRAB technique consists of coating a solid phase with antigen or antibody, reacting the coated solid phase with test serum, and reacting the test serum with a conjugate of avidin and antigen or antibody. The test serum is then reacted with a biotin-labeled indicator enzyme. This assay technique has the same disadvantages as the BRAB method, and, in addition, it is difficult to synthesize and obtain acceptable yields of avidin-antigen or avidin-antibody conjugates. Also, some immunological reactivity of the antigen or antibody is lost upon forming a conjugate with avidin.
In U.S. Pat. No. 4,228,237, another biotin-avidin assay is described in which an enzyme-labeled avidin and a biotin-labeled reagent are utilized. In this assay, a solid phase containing a specific binding substance for a ligand of interest is incubated with a liquid medium suspected of containing the ligand of interest. Next, a biotin-labeled specific binding substance for the ligand and an enzyme-labeled avidin are added. Alternatively, the biotin-labeled specific binding substance for the ligand is bound to the enzyme-labeled avidin. The unreacted reagents are separated from the insoluble phase after incubation, and the enzyme activity of either the insoluble phase or the separated unreacted reagents is determined as a measure of the amount of ligand in the liquid medium.
This method has the same limitations as described above for the BRAB method such as the nonspecific binding of the highly positively-charged avidin. Also, the avidin-enzyme complex is not very stable, particularly when the complex is of a high concentration or at temperatures above 37.degree. C.